This invention relates to sensing. More particularly, the present invention relates to apparatus and methods for sensing refractive index changes by monitoring the energy transfer between a chromophore acceptor and a donor.
Optical sensors, including sensors that monitor absorbance changes and refractive index changes proximate a sensing area are used in the fields of chemical, biochemical, biological or biomedical analysis, process control, pollution detection and control, and other areas. Luminescent (including fluorescent and phosphorescent) markers also find a wide variety of applications in science, medicine and engineering. These sensors and techniques are adaptable to a wide variety of samples including biological samples and extracts (such as physiological fluids, nucleic acid and/or protein-containing solutions, microbial cultures, etc.), environmental samples (such as water sources), industrial, especially chemical reagents, products and wastes, etc.
Surface-plasmon resonance (SPR) is a popular sensing technique in the pharmaceutical industry and biological research. SPR is but one of a large class of optical biosensors collectively referred to as evanescent wave-based detectors. This class includes film waveguide grating couplers, film prism waveguide couplers and long-period grating waveguide couplers. The essential feature of all these techniques is that a standing xe2x80x9cevanescentxe2x80x9d wave is generated above the sensing surface by a short wavelength""s distance from the surface (approximately 100-200 nm) that is sensitive to the local dielectric environment. By changing the local refractive index, the standing wave is altered, requiring either a new angle of incident light to set up the xe2x80x9cresonance conditionxe2x80x9d or inducing a phase shift of the reflected light. Since all proteins, independent of sequence, contribute almost the same refractive index per unit mass, this technique can serve as a mass detector. A linear correlation between resonance angle shift and surface protein concentration has been demonstrated, allowing real time detection of mass change without the need for labeling. All evanescent wave techniques are variations on this essential theme.
One limitation of evanescent wave methods is that they do not readily lend themselves to miniaturization. This limitation makes massive deployment of similar sensing elements on small surfaces extremely problematic. Another limitation of these techniques is that the sensitivity of these optical sensors is limited by many factors such as signal to noise ratio such that the sensitivity of these techniques are usually limited to about 10xe2x88x925 or 10xe2x88x926 Moles/liter in the sensing area.
Another known sensing technique is the monitoring of the energy transfer between a luminescent donor-acceptor pair as a function of the changing distance between the donor-acceptor pair. Luminescent structures are either man-made (see, e.g., Alivisatos, A. P., xe2x80x9cPerspectives on the Physical Chemistry of Semiconductor Nanocrystals,xe2x80x9d Journal of Physical Chemistry, 100, 31, pp. 13226-13239 (1996); Chan, W. C. and Nie, S., xe2x80x9cQuantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection,xe2x80x9d Science, 281, pp. 2016-2018, (1998); Davies, J. H. and Long, A. R. (eds.), Physics of Nanostructures,xe2x80x9d St. Andrews, Institute of Physics Publicshing Ltd., (1992)) or naturally occurring (see, e.g., Glazer, A. N. and Mathies, R. A., xe2x80x9cEnergy-transfer fluorescent reagents for DNA analyses, xe2x80x9cCurrent Opinions in Biotechnology,xe2x80x9d 8 (1), pp. 94-102, (1997); Yardley, J. T., xe2x80x9cIntroduction to Molecular Energy Transfer,xe2x80x9d New York, Academic Press (1980). An example of a man-made structure is a semiconductor sandwich (e.g., GaAs disposed between AlGaAs layers), which shares all of the attributes of a transition dipole structure that both absorbs and emits light and is directional. For more details on these structures, refer to the Chan and Nie and Davies references, the entire contents of which are incorporated herein by reference. These structures, which may be referred to as quantum dots, quantum wires, quantum well structures, or nanocrystals, can be coated to stabilize them in various solutions. More commonly, however, luminescent pairs of donors and acceptors typically include luminescent molecules such as dyes or lanthanides having light absorption and emission properties. For a review of Fluorescent Resonance Energy Transfer, see, Selvin (1994) Fluorescence Resonance Energy Transfer, in Biochemical Spectroscopy, a volume of Methods in Enzymology, Academic Press, Ed. Kenneth Sauer.
U.S. Pat. No. 5,639,615 describes measuring luminescent energy transfer between lanthanide chelate acceptor-donor pairs. The resonance energy transfer described in U.S. Pat. No. 5,639,615 involves detecting the distance between a donor and an acceptor in a portion of a sample by exposing a sample including the donor located at a first position and the acceptor located at a second position to light at a first wavelength capable of inducing a first electronic transition in the donor. The spectral overlap of the donor emission and acceptor absorption is sufficient to enable energy transfer from the donor to the acceptor as measured by a detectable decrease of donor luminescence intensity or a detectable increase in acceptor luminescence intensity. Then the intensity of a first emission of light from the sample portion at a second wavelength is detected, which results from a second electronic transition in the donor. The intensity of the first emission of light correlates with the distance between the first and second positions. In other words, the closer the positions, the greater the energy transfer and the greater the decrease in energy emitted from the donor. An alternative scheme described in U.S. Pat. No. 5,639,615 involves the detection of the intensity of a second emission of light from sample portion at a third wavelength, in which the third wavelength is longer than the first wavelength and results from an electronic transition in the acceptor. The intensity of the second emission of light inversely correlates with the distance between the first and second positions of the sample portion. Thus, the closer the positions, the greater the energy transfer and the greater the acceptor luminescence.
The general method described in U.S. Pat. No. 5,639,615 may be used to measure the static or dynamic distance between two positions, for example, two atoms or molecules. In particular, the method can be used to monitor the status of a polymerase chain reaction. In this instance, the sample portion may include a target nucleic acid strand having a first strand portion and a probe nucleic acid strand labeled proximal to one end with the acceptor and proximal to the other end with the donor. Thus, the donor and the acceptor are separated from each other by the opposite ends of the second strand. The first and second strands are sufficiently complementary to hybridize under annealing conditions. If the second strand is of sufficient length to provide a detectable difference in the aggregate energy transfer from the donor to the acceptor upon hybridization of the first and second strands, as compared with the aggregate energy transfer from the donor to the acceptor when the first and second strand portions are not hybridized, a detectable difference in energy transfer can be measured. The detectable difference is measured as at least one of a detectable decrease or quenching of donor luminescence or detectable increase in acceptor luminescence, and the distance between the acceptor and donor, as a function of changing luminescence, indicates whether the nucleic acid strands have hybridized. Thus, as the reaction proceeds, the stepwise increase in the amount of target nucleic acid is reflected in a stepwise decrease in energy transfer.
The sensitivity of methods using resonance energy transfer as a function of the changing distance between the donor-acceptor pair can be as high as 10xe2x88x9212 Moles/liter. Although these conventional resonance energy transfer techniques have many advantages, they rely on the change in distance between the donor and the acceptor pair to determine the energy transfer between the donor and the acceptor. It would be advantageous to provide a sensor and sensing methods that provide the advantages of optical sensing techniques discussed above and have the sensitivity of the conventional resonance energy transfer techniques. It would also be desirable to provide a sensing technique that facilitates the miniaturization of the sensor and could be deployed on a wide variety of surface and in various environments.
The invention relates to a sensor and methods of sensing. According to one aspect of the invention, a sensor includes a donor for emitting energy and an acceptor for receiving energy. According to this aspect, a sensing area is disposed between the donor and the acceptor, and a photon source for directing photons towards the donor. A detector is also provided for monitoring energy transfer between the acceptor and the donor as a function of the change in refractive index proximate the sensing area. The donor and acceptor can be any luminescent material capable of functioning as an acceptor-donor energy transfer pair. For example, the donor and the acceptor may include dye molecules or atoms, or alternatively, the donor and the acceptor may include a semiconductor crystal, quantum dot, quantum wire or quantum well structure. These terms will be referred to herein as either semiconductor crystal or quantum well structures. In another aspect, the donor and the acceptor may include fluorescent beads or fluorescent proteins, or other luminescent chromophores having a non-zero transition dipole moment. According to one aspect of the invention, the sensor is adapted to detect interactions between biomolecules, or between biomolecules and other chemical entities.
In another aspect of the invention, a method of sensing the refractive index change is provided. According to this aspect, the method includes monitoring the Fxc3x6rster resonance energy transfer between an acceptor and a donor. As with the sensor described above, the acceptor and the donor may include a semiconductor crystal, atom, a quantum well structure, a fluorescent dye, a protein or a fluorescent bead.
Another aspect of the invention pertains to a method of sensing interaction between biomolecules or between biomolecules and other chemical entities. This aspect includes providing an acceptor and donor pair having a sensing area disposed between the pair and providing biomolecules proximate the sensing area. According to this aspect, photon energy is directed towards the donor and the transfer of photon energy between the donor and the acceptor is monitored as a function of refractive index proximate the sensing area.
Still another aspect of the invention relates to biosensor that includes a substrate having a donor and acceptor pair disposed on a surface of the substrate and a sensing area disposed between the donor and the acceptor. According to this aspect, a photon source for directing photon energy towards the donor and means for detecting energy transfer between the donor and the acceptor are provided. This aspect of the invention further includes means for correlating the energy transfer with a change in refractive index proximate the sensing area. In one embodiment of the invention, the biosensor substrate may include a variety of structures that are used in biomolecular analysis. For example, the substrate may a microplate well, a slide including biomolecules attached thereto, or a microfluidics channel.
Another aspect of the invention pertains to a method of manufacturing a sensor. This aspect includes providing an acceptor, a donor and a sensing area disposed between the acceptor and the donor on a surface and directing energy towards the donor. This aspect further includes contacting the sensor with a solution and monitoring the energy transfer between the acceptor and the donor as a function of the change in refractive index of the sensing area.
The invention provides sensing methods and sensors having extremely high sensitivity and the ability to be deployed on a wide variety of surfaces. The sensors and sensing methods of the present invention can be used to sense in a wide variety of environments in which the refractive index of a sensing surface or area changes. For example, the sensing methods and sensors described herein can be used to sense chemical reactions and biomolecular reactions.
Additional advantages of the invention will be set forth in the following detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the invention as claimed.